JPH0813279B2 - Desoxyribonucleic acid sequencing method - Google Patents

Abstract

The method comprises subjecting the deoxyribonucleic acid in a concentration of 10<-16> to 2 x 10<-14> mol per mu l, in the case of double-stranded deoxynucleic acid which is not in the supercoiled form preferably in a concentration of 10<-16> to 3 x 10<-15> mol per mu l, a) to a heat-denaturation step b) to an annealing step and c) to a nucleic acid synthesis step with partial chain termination, repeating steps a) to c) 10 to 60 times and subsequently fractionating the resulting deoxynucleic acid fragment mixture according to length, and determining the sequence from the fragment spectrum. o

Description

FIELD OF THE INVENTION The present invention relates to a method for sequencing desoxyribonucleic acid.

Analysis of the nucleotide sequence of nucleic acids is a central technique in molecular biology.

Prior Art Currently, there are two basic techniques for conventional DNA sequencing. The first is the method of Maxam and Gilbert (Methods Enzymol., Volume 65, 1980).
Year pp. 499-560) and the other one is Sang (Sang
er) method (Proc.Natl.Acad.Sci.USA, Vol. 74, 1977).
Year, page 5463).

Traditionally, most Zanger sequencing methods are used for DNA-sequencing, in which the sequence starts with an oligonucleotide primer complementary to a part of the DNA-strand to be sequenced, In the presence of a mixture of four desoxynucleotide triphosphonates and one didesoxynucleotide triphosphonate, the polymerase produces complementary copies of the chain to be sequenced. At this time, it is important to use didesoxynucleotide triphosphonate. This is because incorporation of this didesoxynucleotide triphosphonate into newly synthesized DNA eliminates the hydroxyl group and chain extension is no longer possible, thus providing sequence-specific chain breaks. This is because it occurs. This chain extension reaction is generally carried out in separate formulations for all four possible mixtures of four desoxynucleotide triphosphates and one didesoxynucleotide triphosphate. The length-dependent separation of the resulting nucleic acid fragments is then carried out by standard electrophoretic methods, which makes it possible to read the nucleotide sequences directly. To distinguish between newly synthesized DNA and DNA to be sequenced, oligonucleotide primers, or one of at least four desoxynucleotides or didesoxynucleotides, such as radioisotopes or recently fluorescent dyes, can be used. Label with.

In all currently most used methods, this reaction is carried out in two or three steps: in the first step (not required in single-stranded-DNA), the complementation of both DNAs to be sequenced The separation of the dynamic chains is carried out at elevated pH or temperature. Then increase the temperature (about 65
At 0 ° C.) an oligonucleotide primer is added at the beginning of the so-called annealing reaction, which primer then binds to the chain to be sequenced in the course of cooling of the solution. In the third step, the original sequencing reaction is performed at a temperature suitable for the polymerase used.

Currently, only three different types of polymerases are used for DNA sequencing: the Klenow-fragment of E. coli polymerase I, the modified form of T7-polymerase and Thermus aquaticu.
s) thermostable Taq-polymerase.

All single-stranded DNA-samples as well as double-stranded DNA-samples present in supercoiled form can be routinely sequenced in the manner currently available. However, the double-stranded DNA-sample present in this supercoiled form should not exceed a critical size (about 20 kilobases). In any case, there is currently no conventionally effective method for sequencing supercoil-DNA using Taq-polymerase. This is particularly desirable, since the higher reaction temperatures possible here eliminate the problems resulting from the formation of secondary structures.

In addition to the above-mentioned limitations, the sequencing of linear double-stranded DNA-molecules is particularly problematic. DNA-molecules of this kind are especially suitable for so-called polymerase chain reaction (PCR) (eg Mulli).
s et al., Methods Enzymol. Volume 155, 1987, 1973-19
(Page 77) final product. Besides its wide applicability in molecular biology research, this sensitive method
Due to the in vitro expansion of the A fragment, especially in the molecular level analysis of genetic diseases, in court identification problems,
And of significant importance in prenatal diagnostics. In all of the above cases, very low amounts of nucleic acid are provided, thus not sufficient for sequencing analysis by conventional methods.

A sequencing method for PCR-fragments based on the Zanger method is described by Saiki et al. (Science, 239, 1988, pages 487-491). In any case, this method relies heavily on the optimal performance of the individual successive reaction steps, making its application to conventional methods difficult. In addition, methods for producing on chemical chain fragments by the Maxam-Gilbert principle have also been described (eg Keohavong
Et al., DNA, Volume 7 (1), 1988, pp. 63-70; Voss et al., Nucl. Acids. Res. Volume 17 (7), 1989, 2517-2.
527). The disadvantage of this method is the need for elaborate chemical reactions following PCR to be carried out with partially toxic substances. Furthermore, in principle, a significantly superior method was reported in which sequence-specific incorporation of thiodesoxynucleotides was carried out during PCR, which was followed by formation of defined chain fragments using an alkylation reaction. (Nakamaye et al., Nucl. Acids Res., Volume 16 (21), 19
88, pp. 9947-9959). However, this method has the serious drawback that only very little signal intensity occurs, limiting the range available for sequencing to 250 bases at the most.

Recently, a method using Taq polymerase and performing a normal reaction cycle a few times has been reported (Levedako
u et al., Bio Techniques, Volume 7, 1989, 438-422.
P .; Carters et al., Bio Techniques, Volume 7, 1989,
Pp. 494-499).

When using fluorescent dyes for the labeling of newly synthesized fragments, it is significant to provide a method which is capable of obtaining many times higher fragment yields than using conventional methods. is required.

Means for Solving the Problems An object of the present invention is a method for sequencing desoxyribonucleic acid, which comprises: a) heat denaturation step, b) annealing step, and c) nucleic acid with partial chain interruption. The steps of a) to c) are repeated 10 to 60 times, the resulting desoxynucleic acid fragment mixture is then separated by length and the sequence determined from this fragment spectrum.

Guide the DNA to steps a) to c) at a concentration of 10 ^{−16 to} 2.10 ⁻¹⁴ molar per μ. In the case of double-stranded, non-supercoiled DNA, the concentration is advantageously in the lower part of the range, ie about 10 ^{−16 to} 3.10 ⁻¹⁵ mol / μ.

The heat denaturation step is carried out at a temperature of 92-98 ° C, preferably 95 ° C.

The annealing process is successfully achieved at temperatures between 35 ° C and 75 ° C.

Starting from the oligonucleotide primer in the nucleic acid synthesis step, this primer must be complementary to a partial region of the single strand to be sequenced. Primers of about 10 to 50 nucleotides, preferably 15 to 30 nucleotides in length, are attached to the DNA to be analyzed. The molar ratio of oligonucleotide primer to sequence to be sequenced is preferably between 5 and 30, ie 5 to 30 moles of primer are used per mole of sequence to be sequenced. This primer should then be sequenced in the presence of a mixture of four desoxynucleotide triphosphates and at least one didesoxynucleotide triphosphate.
It extends complementary to the DNA-strand. This extension is carried out enzymatically with a thermostable polymerase, such as Taku-polymerase. Nucleic acid synthesis process is 65-85 ℃ for Tac-Polymerase
Performed well at temperatures of.

Steps a) to c) are carried out sequentially about 0 to 60 times, preferably 10 to 30 times.

The addition of didesoxynucleotide triphosphonates is done to sequence-specifically interrupt the synthesis of complementary DNA. A particular one of four didesoxynucleotide triphosphonates can be used for chain breaks. In this case the DNA-synthesis has to be carried out four times, i.e. once with each didesoxynucleotide triphosphonate.

It is also possible to use a mixture of four didesoxynucleotide triphosphonates. In this case, the composition is 1
Carry out only once.

In order to recognize the newly synthesized nucleic acid, it must be labeled. This can be done by using labeled oligonucleotide primers or labeled desoxy- or didesoxynucleotide triphosphates. Labeling can be carried out with radioisotopes, enzymes, specific binding sites for detectable molecules or, preferably, fluorescent dyes.

Implementation of the individual steps described above is known (eg, Tabo
r et al., Proc. Natl. Acad. Sci. USA, Vol. 84, 1987, No. 4
767-4771).

The method according to the invention has the following advantages over the currently available DNA sequencing methods: a) PCR-in a reliable and simple way by this method.
The product can be sequenced (example 1), b) this method is for the sequencing of macromolecules, double-stranded DNA-samples such as λ-DNA (example 2) It allows sequencing of double-stranded DNA even in the absence of.

c) The method is also applicable to single-stranded DNA, leading to significantly higher reaction product yields than the conventional method for all DNA-samples, and therefore correspondingly lesser amounts.
The amount of DNA is sufficient for sequencing, and in particular the applicability of fluorescent labeling is no longer limited by the amount of DNA-provided.

For linear double-stranded DNA sequencing, higher fragment yields are of particular importance. This is because reliable sequencing of this type of sample is
This is because it only succeeds when using concentration. The effect of the amount of DNA used for sequencing on the quality of the sequencing reaction is clear from Example 4.

The method according to the present invention and the DNA currently used by Zhanger
The decisive difference between the sequencing methods is that they carry out a very large number of cycles of the sequencing reaction, are cyclical and additionally due to the significantly higher ratio of primer molecules to DNA-molecules to be sequenced Very low DNA concentration. The very high number of cycles on the one hand allows a relatively high fragment yield in the use of a large excess of oligonucleotide primer. In particular, very little DNA-
The combination of concentration and primer and DNA-concentration in a significantly larger ratio gives rise to the possibility to sequence very critical DNA-samples such as double-stranded PCR-products.

EXAMPLE 1 PCR-Sequencing of the product PCR pUC18-derivative 0.1 fMol containing a 600 bp insert at the multi-cloning site (-21) -standard-primer (5'-GGT TGT AAA ACG ACG GCC AGT-
3 ') and reverse-primer (5'-CAC ACA GGA
AAC AGC TAT GAC-3 ') in the presence of 4 pMol each, and in the presence of 20 nMol each of the four desoxynucleotide triphosphates PCR-buffer (50 mM KCl, 10 mM Tris-HCl, 1.
5mM MgCl _2, 0.01% gelatin, pH 8.3) in a volume of 100 microns, Taku - increased polymerase (Cetus) in 5 units.

For this, the reaction mixture was coated with 100 μl of mineral oil and the temperature programmable block (Programmable
Dri-Block PHC-1, Techne) 96 ° C for 1 minute; 37 ° C,
This was followed by 30 temperature cycles consisting of 0.5 min; and 72 ° C., 1.0 min.

Subsequently, Biospin p30-column (Bior
The desoxynucleotide triphosphates were separated after equilibrating the column with PCR-buffer for the aqueous phase of the reaction solution according to the manufacturer's instructions.

Sequencing The eluent from the Biospin column was used for sequencing without further treatment. The following formulations were used for the four sequencing reactions.

Each of the four reaction formulations was subjected to 40 cycles of 96 ° C., 1.0 min; 55 ° C., 0.5 min; and 72 ° C., 1.0 min, similar to the PCR reaction.

Subsequently, the aqueous phases of the four reaction compounds were sequentially treated with 3M NaOAc.
(PH 7.8) Pipette over 20μ. Then, add 500 μl of ethanol at a temperature of −20 ° C. to this solution, mix the solution, and place on dry ice for 15 minutes.
15 minutes at 0 rpm and room temperature, Eppendorf
It was centrifuged in a centrifuge 5415c. The pellet was washed once again with 80% ethanol 800μ, dried in vacuum for 2 minutes, taken up in 50mM EDTA (pH7.5) 2μ and deionized formamide 6μ, heated to 95 ° C for 2 minutes,
Cooled in ice water, DNA-Sequencer (Sequencer) 370A
(Applied Bio-systems) electrophoresis (acrylamide (AA) 6%, AA / bis-AA: 40: 2 PAG; buffer: 0.089M)
Tris, 0.089M borate, 2M EDTA, 7M urea, pH 8.3; Power: 2
0W). Control of the sequencer and evaluation of the electropherogram were performed in the manufacturer's software description 1.30.

The obtained results are shown in FIG. Comparison of the revealed sequences with previously known sequences has shown that at least base 400 can be examined by the method described above, except for the two sites of interest.

Example 2 Sequencing of λ-DNA Sequencing of λ-DNA (λ-ZAP, Stratagene) was performed as in Example 1. PCR-A considerable amount of λ-DNA instead of the product
Was used and all other parameters were retained.

The results are shown in Fig. 2. In this case too, a reliable search for at least 400 bases was carried out in the manner described.

Example 3 Sequencing of linearized plasmid Plasmid pUC18 was linearized with the restriction endonuclease Xmn I and sequenced as in Example 1. The difference from Example 1 is
DNA 0.1 pMol (A- and C-reaction) or 0.2 pMol (G
-And T-reactions), as well as primers 2pMol (A, C) or 4pMol (G, T) and as in the PCR of Example 1, but with only 10 temperature cycles.

The results are shown in Fig. 3. With the exception of the three unrelated bases, it was possible in this experiment to determine at least 400 bases, which is also reliable.

Example 4 Sequencing of PCR-products with different DNA amounts The PCR fragment of base 1450 was sequenced exactly as in Example 1, the amount of DNA used varying between 0.05 and 0.8 pMol. The results obtained for the first 130 bases are shown in FIG. As the amount of DNA increases, the non-specific interruption increases significantly.

[Brief description of drawings]

1 is an electropherogram showing the result of Example 1, FIG. 2 is an electropherogram showing the result of Example 2, and FIG. 3 is a result of Example 3. FIG. 4 is an electropherogram, and FIG. 4 is an electropherogram showing the results of Example 4. In the electropherograms of the drawings, -represents T, --- represents C, --- represents A, ---
-Represents G.

 ─────────────────────────────────────────────────── ─── Continued Front Page (56) References Bio Technologies, 7 (1989) P. 438-442 Bio Technologies, 7 (1989) P. 494-499 Kaoru Fujinaga, "Fundamentals and New Development of Gene Amplification PCR" (Head 2-12-10) Kyoritsu Publishing P. 19-22

Claims (1)

[Claims] 1. A method for sequencing desoxyribonucleic acid, wherein the desoxyribonucleic acid contains 10 ^{−16 to} 2.1 per μl of desoxyribonucleic acid.
In the case of a double-stranded desoxynucleic acid which does not exist in the supercoiled form at a concentration of 0 ^-14 molar, preferably 10 ^-16 ~
3. At a concentration of ^10-15 mol, a) heat denaturation step, b) annealing step, and c) nucleic acid synthesis step with partial chain interruption, and steps a) to c) are repeated 10 to 60 times. A method for sequencing desoxyribonucleic acid, characterized in that the subsequently obtained desoxynucleic acid fragment mixture is separated by length and the sequence is determined from this fragment spectrum.